Azaspiracid Shellfish Poisoning: A Review on the Chemistry, Ecology, and Toxicology with an Emphasis on Human Health Impacts

Azaspiracids (AZA) are polyether marine toxins that accumulate in various shellfish species and have been associated with severe gastrointestinal human intoxications since 1995. This toxin class has since been reported from several countries, including Morocco and much of western Europe. A regulatory limit of 160 μg AZA/kg whole shellfish flesh was established by the EU in order to protect human health; however, in some cases, AZA concentrations far exceed the action level. Herein we discuss recent advances on the chemistry of various AZA analogs, review the ecology of AZAs, including the putative progenitor algal species, collectively interpret the in vitro and in vivo data on the toxicology of AZAs relating to human health issues, and outline the European legislature associated with AZAs

Marine Algal Toxin Azaspiracid Is an Open-State Blocker of hERG Potassium Channels

Azaspiracids (AZA) are polyether marine dinoflagellate toxins that accumulate in shellfish and represent an emerging human health risk. Although human exposure is primarily manifested by severe and protracted diarrhea, this toxin class has been shown to be highly cytotoxic, a teratogen to developing fish, and a possible carcinogen in mice. Until now, AZA's molecular target(s) has not yet been determined. Using three independent methods (voltage clamp, channel binding assay, and thallium flux assay), we have for the first time demonstrated that AZA1, AZA2, and AZA3 each bind to and block the hERG (human ether-à-go-go related gene) potassium channel heterologously expressed in HEK-293 mammalian cells. Inhibition of K+ current for each AZA analogue was concentration-dependent (IC50 value range: 0.64 - 0.84 μM). The mechanism of hERG channel inhibition by AZA1 was investigated further in Xenopus oocytes where it was shown to be an open state-dependent blocker and, using mutant channels, to interact with F656 but not with Y652 within the S6 transmembrane domain that forms the channel's central pore. AZA1, AZA2, and AZA3 were each shown to inhibit [3H]dofetilide binding to the hERG channel and thallium ion flux through the channel (IC50 value range: 2.1 – 6.6 μM). AZA1 did not block K+ current of the closely related EAG1 channel. Collectively, these data suggest that the AZAs physically block the K+ conductance pathway of hERG1 channels by occluding the cytoplasmic mouth of the open pore. Although the concentrations necessary to block hERG channels are relatively high, AZA-induced blockage may prove to contribute to the toxicological properties of the AZAs

Screening and Treatment for Subclinical Hypertensive Heart Disease in Emergency Department Patients With Uncontrolled Blood Pressure: A Cost‐effectiveness Analysis

ObjectivesPoorly controlled hypertension (HTN) is extremely prevalent and, if left unchecked, subclinical hypertensive heart disease (SHHD) may ensue leading to conditions such as heart failure. To address this, we designed a multidisciplinary program to detect and treat SHHD in a high‐risk, predominantly African American community. The primary objective of this study was to determine the cost‐effectiveness of our program.MethodsStudy costs associated with identifying and treating patients with SHHD were calculated and a sensitivity analysis was performed comparing the effect of four parameters on cost estimates. These included prevalence of disease, effectiveness of treatment (regression of SHHD, reversal of left ventricular hypertrophy [LVH], or blood pressure [BP] control as separate measures), echocardiogram costs, and participant time/travel costs. The parent study for this analysis was a single‐center, randomized controlled trial comparing cardiac effects of standard and intense (<120/80 mm Hg) BP goals at 1 year in patients with uncontrolled HTN and SHHD. A total of 149 patients (94% African American) were enrolled, 133 (89%) had SHHD, 123 (93%) of whom were randomized, with 88 (72%) completing the study. Patients were clinically evaluated and medically managed over the course of 1 year with repeated echocardiograms. Costs of these interventions were analyzed and, following standard practices, a cost per quality‐adjusted life‐year (QALY) less than $50,000 was defined as cost‐effective.ResultsTotal costs estimates for the program ranged from$117,044 to $119,319. Cost per QALY was dependent on SHHD prevalence and the measure of effectiveness but not input costs. Cost‐effectiveness (cost per QALY less than$50,000) was achieved when SHHD prevalence exceeded 11.1% for regression of SHHD, 4.7% for reversal of LVH, and 2.9% for achievement of BP control.ConclusionsIn this cohort of predominantly African American patients with uncontrolled HTN, SHHD prevalence was high and screening with treatment was cost‐effective across a range of assumptions. These data suggest that multidisciplinary programs such as this can be a cost‐effective mechanism to mitigate the cardiovascular consequences of HTN in emergency department patients with uncontrolled BP.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136283/1/acem13122.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136283/2/acem13122_am.pd

Comparative analysis of three brevetoxin-associated bottlenose dolphin (Tursiops truncatus) mortality events in the Florida Panhandle region (USA)

In the Florida Panhandle region, bottlenose dolphins (Tursiops truncatus) have been highly susceptible to large-scale unusual mortality events (UMEs) that may have been the result of exposure to blooms of the dinoflagellate Karenia brevis and its neurotoxin, brevetoxin (PbTx). Between 1999 and 2006, three bottlenose dolphin UMEs occurred in the Florida Panhandle region. The primary objective of this study was to determine if these mortality events were due to brevetoxicosis. Analysis of over 850 samples from 105 bottlenose dolphins and associated prey items were analyzed for algal toxins and have provided details on tissue distribution, pathways of trophic transfer, and spatial-temporal trends for each mortality event. In 1999/2000, 152 dolphins died following extensive K. brevis blooms and brevetoxin was detected in 52% of animals tested at concentrations up to 500 ng/g. In 2004, 105 bottlenose dolphins died in the absence of an identifiable K. brevis bloom; however, 100% of the tested animals were positive for brevetoxin at concentrations up to 29,126 ng/mL. Dolphin stomach contents frequently consisted of brevetoxin-contaminated menhaden. In addition, another potentially toxigenic algal species, Pseudo-nitzschia, was present and low levels of the neurotoxin domoic acid (DA) were detected in nearly all tested animals (89%). In 2005/2006, 90 bottlenose dolphins died that were initially coincident with high densities of K. brevis. Most (93%) of the tested animals were positive for brevetoxin at concentrations up to 2,724 ng/mL. No DA was detected in these animals despite the presence of an intense DA-producing Pseudo-nitzschia bloom. In contrast to the absence or very low levels of brevetoxins measured in live dolphins, and those stranding in the absence of a K. brevis bloom, these data, taken together with the absence of any other obvious pathology, provide strong evidence that brevetoxin was the causative agent involved in these bottlenose dolphin mortality events

AZASPIRACIDS – Toxicological Evaluation, Test Methods and Identifcation of the Source Organisms (ASTOX II)

Since the Irish monitoring program was set up in 2001 azaspiracids (AZAs) have been detected in shellfish above the regulatory limit every year with the exception of 2004. The south west coast of Ireland is especially prone to the onsets of AZA events. Over this period a number of poisoning incidents associated with this toxin group have occurred, all related to Irish shellfish. In 2003 the Marine Institute was awarded funding for a research project named ASTOX. This project was very successful in producing a range of reference materials (RMs, which are essential for accurate detection and monitoring, and which up to this point were unavailable. The project also examined the toxicity of AZAs, primarily using in vitro cell assays but some in vivo studies were also performed. The overall aims of the ASTOX 2 project were to strengthen knowledge on the causative organism and toxicity of AZAs. The project aims were grouped into three areas: ecology, chemical support and toxicology.Marine Institute Marine Research Sub Programme (NDP 2007 - 2013), co financed under the European Regional Development Fund

Concurrent Exposure of Bottlenose Dolphins (Tursiops truncatus) to Multiple Algal Toxins in Sarasota Bay, Florida, USA

Sentinel species such as bottlenose dolphins (Tursiops truncatus) can be impacted by large-scale mortality events due to exposure to marine algal toxins. In the Sarasota Bay region (Gulf of Mexico, Florida, USA), the bottlenose dolphin population is frequently exposed to harmful algal blooms (HABs) of Karenia brevis and the neurotoxic brevetoxins (PbTx; BTX) produced by this dinoflagellate. Live dolphins sampled during capture-release health assessments performed in this region tested positive for two HAB toxins; brevetoxin and domoic acid (DA). Over a ten-year study period (2000–2009) we have determined that bottlenose dolphins are exposed to brevetoxin and/or DA on a nearly annual basis (i.e., DA: 2004, 2005, 2006, 2008, 2009; brevetoxin: 2000, 2004, 2005, 2008, 2009) with 36% of all animals testing positive for brevetoxin (n = 118) and 53% positive for DA (n = 83) with several individuals (14%) testing positive for both neurotoxins in at least one tissue/fluid. To date there have been no previously published reports of DA in southwestern Florida marine mammals, however the May 2008 health assessment coincided with a Pseudo-nitzschia pseudodelicatissima bloom that was the likely source of DA observed in seawater and live dolphin samples. Concurrently, both DA and brevetoxin were observed in common prey fish. Although no Pseudo-nitzschia bloom was identified the following year, DA was identified in seawater, fish, sediment, snails, and dolphins. DA concentrations in feces were positively correlated with hematologic parameters including an increase in total white blood cell (p = 0.001) and eosinophil (p<0.001) counts. Our findings demonstrate that dolphins within Sarasota Bay are commonly exposed to two algal toxins, and provide the impetus to further explore the potential long-term impacts on bottlenose dolphin health

Comparative effects of the marine algal toxins azaspiracid-1, -2, and -3 on jurkat t lymphocyte cells

Azaspiracids (AZA) are polyether marine toxins of dinoflagellate origin that accumulate in shellfish and represent an emerging human health risk. Although monitored and regulated in many European and Asian countries, there are no monitoring programs or regulatory requirements in the United States for this toxin group. This did not prove to be a problem until June 2009 when AZAs were identified in US seafood for the first time resulting in human intoxications and further expanding their global distribution. Efforts are now underway in several laboratories to better define the effects and mechanism(s) of action for the AZAs. Our investigations have employed Jurkat T lymphocyte cells as an in vitro model to characterize the toxicological effects of AZA1, AZA2, and AZA3. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that AZA1, AZA2, and AZA3 each elicited a lethal response that was both concentration- and time-dependent, with EC50 values in the sub- to low nanomolar range. On the basis of ECK, comparisons, the order of potency was as follows: AZA2 &amp;gt; AZA3 &amp;gt; AZA1, with toxic equivalence factors (TEFs) relative to AZA1 of 8.3-fold and 4.5-fold greater for AZA2 and AZA3, respectively. Image analysis of exposed cells using Nomarski differential interference contrast (DIC) imaging and fluorescent imaging of cellular actin indicated that the morphological effects of AZA1 on this cell type are unique relative to the effects of AZA2 and AZA3. Collectively, our data support the growing body of evidence suggesting that natural analogues of AZA are highly potent and that they may have multiple molecular targets

Comparative effects of the marine algal toxins azaspiracid-1, -2, and -3 on jurkat t lymphocyte cells

Azaspiracids (AZA) are polyether marine toxins of dinoflagellate origin that accumulate in shellfish and represent an emerging human health risk. Although monitored and regulated in many European and Asian countries, there are no monitoring programs or regulatory requirements in the United States for this toxin group. This did not prove to be a problem until June 2009 when AZAs were identified in US seafood for the first time resulting in human intoxications and further expanding their global distribution. Efforts are now underway in several laboratories to better define the effects and mechanism(s) of action for the AZAs. Our investigations have employed Jurkat T lymphocyte cells as an in vitro model to characterize the toxicological effects of AZA1, AZA2, and AZA3. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that AZA1, AZA2, and AZA3 each elicited a lethal response that was both concentration- and time-dependent, with EC50 values in the sub- to low nanomolar range. On the basis of ECK, comparisons, the order of potency was as follows: AZA2 &amp;gt; AZA3 &amp;gt; AZA1, with toxic equivalence factors (TEFs) relative to AZA1 of 8.3-fold and 4.5-fold greater for AZA2 and AZA3, respectively. Image analysis of exposed cells using Nomarski differential interference contrast (DIC) imaging and fluorescent imaging of cellular actin indicated that the morphological effects of AZA1 on this cell type are unique relative to the effects of AZA2 and AZA3. Collectively, our data support the growing body of evidence suggesting that natural analogues of AZA are highly potent and that they may have multiple molecular targets